This is the third submission for my first Merit Award application with the goal to characterize the effect of a large toxin from Gram negative bacteria and its effect on intestinal epithelial barrier function in vitro and in vivo. Lymphostatin (LS) is encoded by lymphocyte inhibitory factor A (lifA), a large gene present in Gram negative bacteria including Enterohemorrhagic (EHEC), Enteropathogenic E. coli, and Citrobacter rodentium. lifA has been identified as the single gene with the strongest statistical association with EPEC induced diarrhea and hemolytic-uremic syndrome caused by EHEC. lifA encodes for a critical enzymatic activity that have been implicated in microbial pathogenesis: a glucosyltransferase motif at 1.6 kb. We have generated new stable, specific in-frame insertion-deletion mutations inactivating this motif. Our preliminary data suggest that LS is involved in disassembly of epithelial barrier function. It appears that the glycosyltransferase activity is responsible for the dissociation of ZO-1 and occludin from tight junctions (TJs) by preventing activation of Cdc42. Hypothesis 1: LS is critical for the disassembly of proteins forming the TJs, resulting in increased paracellular permeability, promoting bacterial translocation and systemic dissemination. Aim 1: To define the mechanisms by which the lymphostatin glucosyltransferase motif impairs intestinal epithelial barrier function in vitro. Further, the effect of LS on TJs protein members appears to be mediated by causing an imbalance in Rho GTPase activation. We show that the glucosyltransferase is involved in inhibition of the Cdc42 pathway. Hypothesis 2: The glucosyltransferase activity present in LS is critical for the disassembly of TJs components by regulating Rho GTPase Cdc42 pathway. Aim 2: To investigate the effect of C. rodentium lifA glucosyltransferase motif on the small GTPase Cdc42 signaling cascade in vitro. Finally, we plan to test the relevance of lymphostatin glucosyltransferase activity for TJs integrity in an animal model. Hypothesis 3: LS induces disassembly of TJs in vivo, leading to invasion and colonization of mucosa, submucosa and extra-intestinal organs. Aim 3: To investigate the mechanism by which glucosyltransferase motif compromises intestinal epithelial barrier function in vivo. The proposed research project will contribute to our understanding of early pathophysiological events that clarify in how bacteria regulate intestinal epithelial barrier function during enteric infection and gain access to mucosa and submucosa with eventually devastating systemic consequences. PUBLIC HEALTH RELEVANCE: Infection with Gram negative bacteria, like Escherichia coli, is a worldwide health threat resulting in diarrhea with significant morbidity and mortality among children, elderly, and deployed troops. Lymphostatin is a large, novel toxin present in Gram negative bacteria, including pathogenic Escherichia coli strains and Citrobacter rodentium infect the intestine and other organs in humans and mice. Our experimental results suggest that lymphostatin regulates intestinal epithelial barrier function, disrupting its integrity. The current proposal investigates the effect of lymphostatin on specific proteins constituting the intestinal epithelial barrier and their regulating pathways.